Lactic acid bacteria are known to represent strong paradigms for the biochemical diversity in oxygen metabolism, due to their lack of respiratory cytochromes and other hemeproteins. Instead these organisms rely on networks of unusual flavin-linked respiratory enzymes, and this application is focused on detailed structural and mechanistic studies of three particularly interesting representatives of this group: (1) NADH oxidase (Nox; 2NADH + O2 to 2NAD+ + 2H2O) from the severe human pathogen Streptococcus pyogenes, (2) NADH peroxidase (Npx; NADH + H2O2 - to NAD+ + 2H2O) from Enterococcus faecalis, and (3) alpha-glycerophosphate oxidase (GlpO; alpha-glycerophosphate + O2 - to dihydroxyacetone phosphate + H2O2) from Streptococcus sp. The GlpO sequence is closely related to those of the membrane-associated a-glycerophosphate dehydrogenases (GlpDs); the enzymes are distinguished primarily by the presence of a 50-residue insert unique to GlpO, which has furthermore been shown in limited proteolysis experiments to represent a flexible surface region that plays a crucial role in flavin reduction. Elucidation of the GlpO structure should provide insight into the structures of the related GIpDs as well and should better define the role of the GlpO surface loop during aglycerophosphate oxidation. While the structural homology between Nox and Npx has been reinforced dramatically with new crystal structures during the current project period, only recently has a third member of this small class of NAD(P)H-linked peroxide/disulfide reductases been identified - the FAD-dependent coenzyme A-disulfide reductase (CoADR; NADPH + CoASSCoA to NADP+ + 2CoASH) from the serious human pathogen Staphylococcus aureus. CoADR nicely complements this emerging group of flavoproteins which utilize a single active-site Cys as the basis for an essential redox center - the Cys-sulfenic acid (Cys-SOH) documented with Nox and Npx, and the Cys-SSCoA disulfide present in CoADR. An additional long-term objective of this application is the ongoing definition of the structural, redox, and mechanistic parameters for Cys-SOH function in Nox and Npx, as these enzymes provide the benchmarks by which protein-SOH centers now known to participate in diverse aspects of redox signaling and catalysis are analyzed. The new structures available for Nox and Npx also provide the basis for continuing analyses of the functional distinction between the relatively rare reduction of O2 to 2H2O catalyzed by Nox and the peroxidatic reaction of Npx. The interpretable electron density map recently calculated for CoADR now makes it possible to model and refine the atomic structure of this enzyme as well, thus allowing detailed comparisons with Nox and Npx and providing structural insights into the basis for the functional asymmetry exhibited by this unusual disulfide reductase.